Further synthetic and biological studies on vitamin D hormone antagonists based on C24-alkylation and C2α-functionalization of 25-dehydro-1α- hydroxyvitamin D3-26,23-lactones

Article abstract of DOI:10.1021/jm060797q

An efficient synthesis and the biological evaluation of 80 novel analogs of 25-dehydro-1α-hydroxyvitamin D₃-26,23S-lactone 2 (TEI-9647) and its 23R epimer (3) in which the lactone ring was systematically functionalized by introduction of a C₁ to C₄ primary alkyl group at the C24 position (5 sets of 4 diastereomers), together with their C2α-methyl, 3-hydroxypropyl, and 3-hydroxypropoxy-substituted derivatives were described. The triene structure of the vitamin D₃ was constructed using palladium-catalyzed alkenylative cyclization of the A-ring precursor enyne with the CD-ring counterpart bromoolefin having the C24-alkylated lactone moiety on the side chain. The CD-ring precursors having 23,24-cis lactones were prepared by using a chromium-mediated syn-selective allylation-lactonization process, and the 23,24-trans lactone derivatives were derived from these via inversion of the C23 stereochemistry. The biological evaluation revealed that both binding affinity for chick vitamin D hormone receptor and antagonistic activity (inhibition of vitamin D hormone induced HL-60 cell differentiation) were affected by the orientation and chain-length of the primary alkyl group on the lactone ring. Furthermore, the C2α-functionalization of the C24-alkylated vitamin D₃ lactones dramatically enhanced their biological activities. The most potent compound to emerge, (23S,24S)-2α-(3-hydroxypropoxy)-24-propyl exhibited almost 1000-fold stronger antagonistic activity (IC₅₀ = 7.4 pM) than 2 (IC₅₀ = 6.3 nM).

Full text of DOI:10.1021/jm060797q

J. Med. Chem. 2006, 49, 7063-7075
7063
Further Synthetic and Biological Studies on Vitamin D Hormone Antagonists Based on
C24-Alkylation and C2r-Functionalization of 25-Dehydro-1r-hydroxyvitamin D3-26,23-lactones
Nozomi Saito,† Toshihiro Matsunaga,† Hiroshi Saito,‡ Miyuki Anzai,‡ Kazuya Takenouchi,‡ Daishiro Miura,‡
Jun-ichi Namekawa,‡ Seiichi Ishizuka,‡ and Atsushi Kittaka*,†
Faculty of Pharmaceutical Sciences, Teikyo UniVersity, Sagamihara, Kanagawa 199-0195, Japan, and Teijin Institute for Bio-Medical
Research, Tokyo 191-8512, Japan
ReceiVed July 7, 2006
An efficient synthesis and the biological evaluation of 80 novel analogs of 25-dehydro-1R-hydroxyvitamin
D3-26,23S-lactone 2 (TEI-9647) and its 23R epimer (3) in which the lactone ring was systematically
functionalized by introduction of a C1 to C4 primary alkyl group at the C24 position (5 sets of 4 diastereomers),
together with their C2R-methyl, 3-hydroxypropyl, and 3-hydroxypropoxy-substituted derivatives were
described. The triene structure of the vitamin D3 was constructed using palladium-catalyzed alkenylative
cyclization of the A-ring precursor enyne with the CD-ring counterpart bromoolefin having the C24-alkylated
lactone moiety on the side chain. The CD-ring precursors having 23,24-cis lactones were prepared by using
a chromium-mediated syn-selective allylation-lactonization process, and the 23,24-trans lactone derivatives
were derived from these via inversion of the C23 stereochemistry. The biological evaluation revealed
that both binding affinity for chick vitamin D hormone receptor and antagonistic activity (inhibition of
vitamin D hormone induced HL-60 cell differentiation) were affected by the orientation and chain-length of
the primary alkyl group on the lactone ring. Furthermore, the C2R-functionalization of the C24-alkylated
vitamin D3 lactones dramatically enhanced their biological activities. The most potent compound to emerge,
(23S,24S)-2R-(3-hydroxypropoxy)-24-propyl exhibited almost 1000-fold stronger antagonistic activity (IC50
) 7.4 pM) than 2 (IC50 ) 6.3 nM).
Introduction
The seco-steroidal hormone 1R,25-dihydroxyvitamin D3
(Figure 1, 1) is the most potent natural metabolite of vitamin
D3 and shows a broad spectrum of biological activities. The
most prominent physiological role of 1 is the regulation of
calcium and phosphorus metabolism as well as bone remodeling
via its action in the bone, intestine, and kidney. Moreover, 1
affects the proliferation and differentiation of various types of
tumor cells and also regulates immune reactions.1,2 The natural
hormone 1 exerts its biological effects through the interaction
with a vitamin D receptor (VDRa), which is a member of the
nuclear receptor superfamily and acts as a ligand-dependent gene
transcription factor with coactivators.3,4 The first step in the
VDR-mediated transactivation is a ligand-binding process to
the ligand-binding domain (LBD) of the apo form of VDR. Next,
the ligand-VDR complex changes conformation into a tran-
scriptionally active holo form, which binds to the coactivators
to activate the target gene.5 During conformational change, helix
12, which is the most C-terminal R-helix of VDR and has the
site for interaction with other proteins such as coactivators, is
important and controls whether the function of a ligand is
agonism or antagonism.6,7
Figure 1. Structures of 1R,25-dihydroxyvitamin D3 (1) and its
representative C2R-modified analogs (1a-c).
than the natural 1, numerous analogs of 1 have been developed.
Although more than two thousands vitamin D analogs have been
synthesized over the past few decades, most of the synthetic
studies of the vitamin D3 analogs have involved side-chain
modification.2,8 On the other hand, we have developed the first
systematic synthesis of novel analogs of vitamin D3 based on
the structural modification of the A-ring core to investigate
A-ring conformation and structure-activity relationships.9-14
During the course of our studies, we found out some function-
alization of C2R position on the A-ring increased the binding
affinity for VDR with potent agonistic activity. Namely,
introduction of methyl (1a),9 3-hydroxypropyl (1b),10 and
3-hydroxypropoxy (1c)11 groups into the C2R position showed
2- to 4-fold higher binding affinity relative to the natural hor-
mone 1. The molecular modeling of the three analogs (1a-c)
based on Moras’ X-ray crystallographic analysis of VDR-1
complex15 showed the analogs (1a-c) fit well to the cavity of
the LBD of the VDR and each C2R substituent on the A-ring
interacted with some amino acid residues of the LBD.10b,11,16
That is, the C2R methyl group of 1a interacts with some
hydrophobic amino acid residues Leu233, Tyr236, and Phe150
in the LBD.16 In the case of 1b and 1c, each C2R terminal
The major reason for therapeutic limitation of 1 is calcemic
and phosphatemic activities. Thus, 1 can cause serious side
effects such as hypercalcemia and hyperphosphatemia at su-
perphysiological levels. Therefore, to find the new vitamin D
analogs, which are more efficacious, safer, and more selective
* To whom correspondence should be addressed. Phone and Fax: (+81)-
42-685-3713. E-mail: akittaka@pharm.teikyo-u.ac.jp.
† Teikyo University.
‡ Teijin Institute for Bio-Medical Research.
a VDR, vitamin D receptor; LBD, ligand-binding domain; NBT, nitro
blue tetrazolium; FCS, foetal calf serum; TPA, 12-O-tetradecanoylphorbol-
13-acetate; PBS, phosphate-buffered saline.
10.1021/jm060797q CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/03/2006

7064 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Saito et al.
Figure 3. Plan for the functionalization of 25-dehydro-1R-hydroxy-
vitamin D3-26,23-lactones.
Figure 2. Structures of 25-dehydro-1R-hydroxyvitamin D3-26,23-
lactones (2 and 3), their C2R-functionalized analogs (2a-c and
3a-c), and 24,24-disubstituted vitamin D3-26,23-lactones (4-7).
Scheme 1. Retrosynthesis
hydroxy group forms a new hydrogen bond between the hydroxy
group and Arg274.10b,11 Such hydrophilic or hydrophobic inter-
action between the C2R side chain and the amino acid residues
in the LBD could improve the binding affinity for the VDR.
In 1999, the first vitamin D antagonists, 25-dehydro-1R-
hydroxyvitamin D3-26,23-lactones, 2 (TEI-9647) and its (23R)-
epimer 3 (TEI-9648), were discovered during the course of
studies on the side-chain modification of the 1R,25-dihydroxy-
vitamin D3-26,23-lactone metabolite17 derived from 1 (Figure
2).18-20 Both vitamin D3 analogs 2 and 3 are the first specific
antagonists of VDR-mediated genomic action of 1.21 Namely,
2 and 3 inhibit the differentiation of human leukemia cells
(HL-60 cells) induced by 1.18a Moreover, 2 suppresses the gene
expression of 25-hydroxyvitamin D3-24-hydroxylase in human
osteosarcoma cells18b and in HL-60 cells18d induced by 1.
Furthermore, 2 antagonizes the genomic-mediated calcium
metabolism regulated by 1 in vivo in rat.18e Vitamin D
antagonists have received considerable attention because of their
possibility to be potential agents for some diseases caused by
the hypersensitivity of the VDR to 1R,25-dihydroxyvitamin D3
(1), such as Paget’s disease of bone,22 which is the most flagrant
example of disordered bone remodeling and the second most
common bone disease after osteoporosis in Anglo-Saxons.22a
Recent studies on Paget’s disease suggested a specific increase
in osteoclasts sensitivity to the differentiation activity of 1 as
the principal mechanism for abnormal bone formation.22,23 With
this background, we set out to conduct an investigation of the
structure-activity relationships of the vitamin D3 lactones from
the standpoint of searching for more potential antivitamin D
molecules, and we found some pertinent modifications of 2 and
3 that resulted in an enhancement of their activities.24 That is,
introduction of the above three motifs, that is, the methyl, the
3-hydroxypropyl, or the 3-hydroxypropoxy groups into the C2R
position of 2 and 3, increased the antagonistic activity up to
30-fold in the case of 2b.24a,25 We also synthesized the 24,24-
dimethylvitamin D3 lactones (4 and 5) and 24,24-ethanovita-
min D3 lactones (6 and 7), which do not have an extra chiral
center on the lactone ring, to examine the effects of the C24
substituents on the biological activity.24b,c The biological evalu-
ation of the 24,24-disubstituted analogs (4-7) revealed that both
the VDR binding affinity and the antagonistic activity were
affected by the substituents and the stereochemistry on the C23
position. Namely, both the (23S)- and (23R)-24,24-dimethyl-
vitamin D3 lactones (4 and 5) had enhanced biological activities
compared to the corresponding 2 and 3, respectively.24b On the
other hand, both the VDR binding affinity and the antagonistic
activity of the (23S)-24,24-ethanovitamin D3 analogs (6) were
improved, however, the biological activities of its stereoisomer
(7) were weaker than those of the original 3.24c
Our previous results as above indicated that the functional-
ization of the C2R and C24 positions can be effective in the
enhancement of the biological activity of the vitamin D3
lactones, and we decided to investigate the further structure-
activity relationships of the vitamin D3 lactones along the C2R
and C24-functionalization strategy for the creation of more
potent vitamin D antagonists applicable to the treatment of
Paget’s disease. Now we designed C24 monoalkylated vitamin
D3 26,23-lactones and their C2R-modified derivatives (Figure
3, 8). That is, we planned the systematic introduction of the
primary alkyl group (C1 to C4 unit) into the C24 position on
the lactone ring (9-12) to examine the influence of both the
alkyl chain length and the stereochemistry of the lactone moiety
on the biological activities. Moreover, we expected both the
VDR binding affinity and the antagonistic activity of the C24
alkylated lactones (9-12) to be enhanced by introducing the
above three motifs, that is, 2R-methyl, 2R-(3-hydroxypropyl),
and 2R-(3-hydroxypropoxy) groups as in above-mentioned our
previous results.24b,c Here, we describe our detailed results of
the systematic structural evolution of the vitamin D3-26,23-
lactones based on the C24-monoalkylation and C2R-modifica-
tion strategy.26
Results
Our synthetic plan of functionalized vitamin D3-26,23-
lactones is shown in Scheme 1. The triene skeleton of the desired
vitamin D3 lactone analogs (8) would be constructed by Trost’s
Pd-catalyzed alkenylative cyclization27 of A-ring precursor
enynes (14 or 14a-c) with the CD-ring bromoolefin counterpart

Synthetic and Biological Studies on Vitamin D Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7065
Scheme 2. Synthesis of 23,24-cis-Substituted Lactone
Derivatives
produce the corresponding lactone derivatives 22-26 and
27-31, respectively. Some of the lactone derivatives were
separated by recycle HPLC (see Supporting Information).
The stereochemistries on the C23 and C24 positions (based
on the steroidal numbering) of each lactone derivative (22-26
and 27-31) were determined by the combination of NOE
experiments and modified Mosher’s method reported by
Kusumi.29 First of all, NOE experiments were carried out to
determine the relative stereochemistries on C23 and C24
positions on the 5-membered lactone ring, and the NOEs
between 23-H and 24-H were observed as shown in Table 1.
Therefore, the stereochemistries of substituents on the lactone
ring of all compounds (22-26 and 27-31) were determined to
be cis-orientation.
Next, the lactone derivatives (22-26) were reduced to the
corresponding diols followed by pivaloylation of the primary
alcohols to give 32-36, which reacted with (R)- or (S)-MTPA
chloride to give the corresponding (S)-MTPA esters (37a-41a)
and (R)-MTPA esters (37b-41b), respectively (Scheme 3). The
values of ∆δ ) δ(S)-MTPA ester - δ(R)-MTPA ester in the 600 MHz
1H NMR spectra of 37-41 were calculated as shown in Figure
4. These data were considered by applying the modified
Mosher’s method, and the configuration at the C23 position of
37-41 was determined to be 23S. From these results and NOE
experiments shown in Table 1, the absolute configuration at
the C24 position of 37-41 was determined to be 24S.
On the other hand, the lactone derivatives (27-31) were also
transformed into the corresponding (S)-MTPA esters (47a-51a)
and (R)-MTPA esters (47b-51b) in good yields (Scheme 4).
Table 1. NOE Experiments on 22-26 and 27-31
compd
23-H to 24-H (%)
24-H to 23-H (%)
22
23
24
25
26
6.09
7.24
8.22
8.29
8.00
5.50
7.47
7.28
5.63
8.03
Similar to the lactones (22-26), the values of ∆δ ) δ(S)-MTPA
1
ester - δ(R)-MTPA
in the H NMR spectra of 47-51 were
ester
calculated, and the data were considered by applying the
modified Mosher’s method. As the result, the absolute config-
uration at the C23 position of 47-51 was determined to be 23R.
From these results and NOE experiments shown in Table 1,
the stereochemistry at the C24 position of 47-51 was deter-
mined to be 24R (Figure 5).
27
28
29
30
31
8.35
8.78
9.55
9.63
9.59
6.98
4.19
8.01
7.98
8.11
The CD-ring precursors (57-61) having 23,24-trans lactone
were synthesized from 23,24-cis lactones (22-26; Scheme 5).
The secondary alcohol derivatives (32-36), which were ob-
tained by the above DIBAL-H reduction of the corresponding
lactones (22-26), were oxidized by TPAP-NMO to produce
the corresponding ketone derivatives (52-56). The ketones
(52-56) were treated with LiAlH(Ot-Bu)3, followed by depro-
tection of pivaloyl groups using DIBAL-H to give the diols (for
stereoselectivity at C23, see Supporting Information No. 2). The
resulting diols were oxidized to produce the desired (23R,24S)-
lactone derivatives, respectively (57-61).
having a C24-alkylated R-methylene-γ-lactone side chain (13).
The alkyl chain substituted lactone ring of 13 could be
synthesized through the stereoselective allylation of the aldehyde
15 by allylic metal species 16.
1. Synthesis and Biological Evaluation of 24-Alkylated
Vitamin D3-26,23-lactones. 1.1. Preparation of CD-Ring
Precursors. We synthesized the CD-ring counterparts, which
have 23,24-cis lactone moiety, using Oshima’s chromium-
promoted syn-selective allylation (Scheme 2).28 The aldehyde
15, which was prepared via oxidative degradation of vitamin
D2,24a reacted with allylic bromides (17-21) in the presence of
low-valent Cr complex generated from CrCl3 and LiAlH4 to
The other 23,24-trans lactones (67-71) were similarly
derived from the corresponding (23R,23R)-alcohol derivatives
Scheme 3. Transformation of 22-26 into the Corresponding (S)- or (R)-MTPA Esters (37a-41a and 37b-41b)

7066 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Saito et al.
Figure 4. Determination of stereochemistry at the C23 position of 37-41 by modified Mosher’s method.
Figure 5. Determination of stereochemistry at the C23 position of 47-51 by modified Mosher’s method.
Scheme 4. Transformation of 27-31 into the Corresponding (S)- or (R)-MTPA Esters (37a-41a and 37b-41b)
(42-46; Scheme 6). The alcohol (42-46) were oxidized by
using TPAP-NMO to give the corresponding ketones (62-66).
Then, the ketones were transformed into the (23S,24R)-lactones
(67-71) through reduction of the C23-keto group, followed by
oxidative lactonization process (method A for 69-71, method
B for 67 and 68).
catalyzed coupling reaction of CD-ring precursors (22-31,
57-61, or 67-71) with A-ring enyne (14) and then deprotection
of the silyl groups under acidic conditions gave the correspond-
ing 24-alkylated vitamin D3-26,23-lactones (72-91), respec-
tively (Scheme 7).
1.3. Biological Activities of 24-Alkylated Vitamin D3-26,-
23-lactones. Biological evaluation of the C24-alkylated vitamin
D3 lactones (72-91) showed that both VDR binding affinity
1.2. Synthesis of 24-Alkylated Vitamin D3-26,23-lactones.
Construction of vitamin D3 triene skeleton was achieved by Pd-
Scheme 5. Preparation of (23R,24S)-Lactones from (23S,24S)-Lactones
Scheme 6. Synthesis of (23S,24R)-Lactones from (23R,24R)-Lactones.

Synthetic and Biological Studies on Vitamin D Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7067
Scheme 7. Synthesis of 24-Alkylated Vitamin D3-26,23-lactones
Table 2. Biological Activities of C24-Alkylated Vitamin D3 Lactones
(72-91)
and their activities relative to 2 were calculated based on the
IC50 values. The antagonistic activities of (23S,24S)-isomers (72,
73, 75, and 76) were enhanced to be 2.2-, 3.5-, 1.8-, and 1.7-
fold higher potencies than that of 2, respectively. In this
(23S,24S)-series, only the vitamin D3 lactone having the propyl
group (74) showed weaker activity than 2. In the case of the
(23S,24R)-series (77-81), the antagonistic activity was increased
by the introduction of the methyl group into the lactone ring
(77) to 2.5-times higher than that of 2. The other analogs
(78-81) showed the weaker potency than 2. On the other hand,
the antagonistic activities of (23R,24S)-isomers (82-86) were
little affected by C24-alkylation, whereas (23R,24R)-lactones
(87-91) showed stronger antagonistic activities than 3. Espe-
cially (23R,24R)-24-propylvitamin D3 lactone (89), which
exerted 5.4-fold higher antagonistic activity than 3. Among these
(23R,24R)-isomers, the antagonistic activity of 24-isobutyl
analog (91) was almost same as that of 3.
VDR
relative
antagonistic
activityb
VDR
relative
antagonistic
activityb
binding
binding
compd
affinitya
compd
affinitya
2
12.3
37.0
166.7
100
1169
284
3
7.2
17.5
0.72
7
16
<0.3
4c
6d
5c
7d
72
73
74
75
76
28.6
30.3
43.5
45.5
27.8
220
345
86
179
174
82
83
84
85
86
11.9
6.5
4.4
7.5
0.97
7
6
8
4
5
77
78
79
80
81
21.7
19.2
8.6
16.7
3.0
250
70
60
58
40
87
88
89
90
91
4.9
7.1
8.8
5.2
1.5
18
13
38
17
9
a The potency of the natural hormone 1 is normalized to 100. b The
potency of 2 (IC50 ) 6.0∼11.0 nM) is normalized to 100 (see Experimental
Section). c See ref 24b. d See ref 24c.
2. Double Modification of C2R- and C24-Positions of
Vitamin D3 Lactones. 2.1. Synthesis of 2R,24-double modi-
fied vitamin D3-26,23-lactones. Next, we focused on the
C2R-modification of the twenty 24-alkylvitamin D3 lactones
(72-91). According to our results, C2R-modification of the
24,24-disubstituted vitamin D3 lactone analogs effectively
enhanced both binding affinity for VDR and antagonistic
activity.24b,c Therefore, we expected that introducing the three
motifs, for example, methyl, 3-hydroxypropyl, and 3-hydroxy-
propoxy groups into the C2R-position of 72-91 would in-
crease the receptor binding affinity and improve the antago-
nistic activity. The C2R-modified 24-alkylvitamin D3 lactones
(2R-methyl, 72a-91a; 2R-(3-hydroxypropyl), 72b-91b; and
2R-(3-hydroxypropoxy), 72c-91c) were similarly synthesized
from the corresponding CD-ring unit 22-26, 67-71, 57-61,
and 27-31 with the A-ring counterpart 14a,32 14b24b and 14c,11b
respectively (Scheme 8). The coupling yields are summarized
in Table 3.
and antagonistic activity were markedly affected by the structure
of the lactone ring, including length of the alkyl chain and the
stereochemistries on C23 and C24 positions (Table 2). We also
show the data of 24,24-disubstituted vitamin D3 lactones
(4-7) for comparison.24b,c The binding affinity for chick
intestinal VDR was examined as described previously,30 and 2
and 3 showed 8 (12.3%) and 14 (7.2%) times weaker potencies
than that of the natural hormone 1. The VDR binding affinity
of the (23S,24S)-24-alkylated vitamin D3 lactones (72-76)
increased to 2.3- ∼ 3.7-fold more potent than that of 2. As the
carbon number of the alkyl group increased, the binding affinity
was also getting higher except for 24-isobutyl analog (76). In
the case of the (23S,24R)-series (77-81), the binding affinity
for VDR seemed to be relatively affected by the alkyl chain
length. That is, the analogs, which have methyl (77), ethyl (78),
and butyl (80) groups, showed slightly higher binding affinity
than 2, and the VDR binding affinities of 24-propyl (79) and
4-isobutyl (81) substituted analogs of 2 decreased to 1.4 and
4.1 times lower than that of 2. On the other hand, the VDR
binding affinity of the (23R)-vitamin D3 lactone was little
affected by introduced alkyl groups (82-91) without regard to
the stereochemistry of the C24 position.
2.2. Biological Activities of Double Modified Vitamin D3
Lactone Derivatives. The biological activities of C2R and C24
double modified analogs are shown in Table 4 (23S-series) and
Table 5 (23R-series), including the previous data of C2R-
functionalized vitamin D3 lactones (2a-c and 3a-c) for
comparison.24a The biological evaluation of the (23S)-series
(72a-81a, 72b-81b, and 72c-81c) demonstrated that the C2R-
modification was remarkably effective in improving the bio-
logical activities of (23S)-24-alkylvitamin D3-26,23-lactones
Next, the antagonistic activities of 72-91 were assessed by
the NBT-reduction method31 in terms of IC50 for differentiation
of HL-60 cells induced by 10 nM of the natural hormone 1,

7068 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Saito et al.
Table 3. Synthetic Yields of C2R-Modified C24-Alkylvitamin D3 Lactones (72a-91a, 72b-91b, and 72c-91c)
(23S)-series
(23R)-series
compd
R1
R2
yield (%)
compd
R1
R2
yield (%)
72a
72b
72c
73a
73b
73c
74a
74b
74c
75a
75b
75c
76a
76b
76c
(24S)-Me
Me
68
61
41
56
62
61
59
51
54
57
52
56
49
59
56
82a
82b
82c
83a
83b
83c
84a
84b
84c
85a
85b
85c
86a
86b
86c
(24S)-Me
Me
53
26
52
67
53
61
54
56
53
50
40
44
49
44
50
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(24S)-Et
(24S)-Pr
(24S)-Bu
(24S)-i-Bu
(24S)-Et
(24S)-Pr
(24S)-Bu
(24S)-i-Bu
(CH2)3OH
O(CH2)3OH
(CH2)3OH
O(CH2)3OH
77a
77b
77c
78a
78b
78c
79a
79b
79c
80a
80b
80c
81a
81b
81c
(24R)-Me
(24R)-Et
(24R)-Pr
(24R)-Bu
(24R)-i-Bu
Me
71
54
54
53
62
68
59
48
57
51
54
51
54
57
43
87a
87b
87c
88a
88b
88c
89a
89b
89c
90a
90b
90c
91a
91b
91c
(24R)-Me
(24R)-Et
(24R)-Pr
(24R)-Bu
(24R)-i-Bu
Me
55
66
51
63
54
58
48
43
50
66
52
57
53
49
47
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
Me
(CH2)3OH
O(CH2)3OH
(CH2)3OH
O(CH2)3OH
Scheme 8. Synthesis of 2R-Modified 24-Monoalkylvitamin
D3-26,23-lactones (72a-91a, 72b-91b, and 72c-91c).
The biological activities of C2R-modified (23R)-type analogs
(82a-91a, 82b-91b, and 82c-91c) were summarized in Table
5. In most of the C2R-modified analogs, enhancement of
the binding affinity for VDR was observed. Especially, the
(23R,24S)-2R,24-dimethyl analog (82b) showed 5.3 times higher
VDR binding affinity than that of 3 and 3.2 times higher VDR
binding affinity than the 2R-nonsubstituted analog (82). It was
also demonstrated that the antagonistic activity of (23R)-24-
alkylvitamin D3 lactones (82-91) was improved by the C2R-
functionalization. However, 82b, 85a, and 86a showed almost
same or lower antagonistic activity relative to the corresponding
C2R-nonsubstituted analogs (82, 85, and 86). In the (24S)-series,
the analogs 82a, 84b, 84c, 85b, and 85c showed more than 10-
fold higher antagonistic activity than 3. In particular, the
antagonistic activity of (23R,24S)-2R-(3-hydroxypropyl)-24-
propylvitamin D3 (84b) was enhanced to 82 times higher than
that of 3. In the case of the (24R)-isomers, 87a, 88a, 89a-c,
and 90a exhibited more than 10 times greater antagonistic
activity than 3, and the most potent (23R,24R)-analog was
2R,24-dimethylvitamin D3 lactone (87a), which showed 68-fold
stronger antagonistic activity than 3 and 26-fold higher antago-
nistic activity than the corresponding C2R-nonsubstituted analog
87.
(72-76 and 77-81; Table 4). Namely, the binding affinity of
(24S)-series (72a-76a, 72b-76b, and 72c-76c) significantly
increased to 1.2-6.8 times higher as compared to 2. On the
other hand, the (24R)-series (77a-81a, 77b-81b, and
77c-81c) showed lower VDR binding affinity relative to
the corresponding (24S)-isomers. However, the affinity for
VDR of some analogs (77a-c, 78a, 79b, and 80a-c) were
slightly improved by the C2R-functionalization up to 3.4
times higher than that of 2. The antagonistic activity of the
all (23S)-type analogs (72-81) was enhanced by introducing
C2R-substituents. That is, all of the (24S)-analogs (72a-76a,
72b-76b, and 72c-76c) showed high antagonistic activity, at
least more than 5.2-fold higher potency (72b). In particular,
(23S,24S)-2R-(3-hydroxypropoxy)-24-propylvitamin D3 lac-
tone (74c) exerted 851-fold stronger antagonistic activity
(IC50 ) 7.4 pM) than 2 (IC50 ) 6.3 nM). On the other hand,
C2R-functionalization of (24R)-series (77a-81a, 77b-81b,
and 77c-81c) also exhibited improvement of the antago-
nistic activity than the corresponding C2R-nonsubstituted
analogs (77-81). Especially, the antagonistic activity of (23S,-
24R)-2-(3-hydroxypropoxy)-24-propylvitamin D3 lactone (79c)
was increased to 121 times stronger (IC50 ) 52 pM) than that
of 2 (IC50 ) 6.3 nM).
Discussion
Plausible Mechanism of VDR Antagonism by Vitamin D3
Lactones. The mechanism of VDR antagonism by 2 is not clear
yet. However, it was found that the complex of 2 and VDR
changed into an unusual transcriptionally inactive form, when
2 binds to the LBD of a VDR.33 Recently, it was revealed that
the two cysteines, Cys403 and Cys410, play an important role
in the VDR antagonism of 2.34 Furthermore, the exo-methylene
lactone structure is indispensable for the antagonistic action of
the vitamin D3 lactones.35 Based on these results, we consider
that the nucleophilic thiol groups of the cysteines could attack

Synthetic and Biological Studies on Vitamin D Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7069
Table 4. Biological Activities of 2R-Modified (23S)-24-Alkylvitamin
D3 Lactones (72a-81a, 72b-81b, and 72c-81c)
VDR
relative
antagonistic
activityb
VDR
relative
antagonistic
activityb
binding
binding
compd
affinitya
compd
affinitya
2ac
2bc
2cc
15.6
18.2
16.4
1019
2989
1160
72a
72b
72c
73a
73b
73c
74a
74b
74c
75a
75b
75c
76a
76b
76c
62.5
83.3
50.0
41.6
34.5
15.2
41.6
58.8
37.0
58.8
66.7
50.0
41.6
40.0
30.3
3752
517
1968
1288
442
77a
77b
77c
78a
78b
78c
79a
79b
79c
80a
80b
80c
81a
81b
81c
22.7
32.3
41.7
25.0
5.64
3.13
8.62
17.9
9.43
21.7
16.4
20.0
4.06
12.7
9.00
6191
1512
1743
248
866
158
2032
913
12115
216
122
205
Figure 6. Structures of VDR antagonists 92 and 93 with presumably
different antagonism on VDR.
600
Conclusions
1537
4200
85135
2913
1158
1660
1219
989
We have systematically synthesized novel vitamin D antago-
nists, 1R-hydroxyvitamin D3-26,23-lactone analogs that have
24-methyl, 24-ethyl, 24-propyl, 24-butyl, and 24-isobutyl groups
on the lactone ring, to investigate the structure-activity relation-
ships on the lactone ring core. As the results demonstrated, both
binding affinity for VDR and antagonistic activity were affected
by the orientation and chain-length of the alkyl group on the
lactone ring. Furthermore, it was also found that the C2R-
functionalization of the C24-alkylated vitamin D3 lactones
remarkably enhanced their biological activities. In particular,
(23S,24S)-2R-(3-hydroxypropoxy)-24-propylvitamin D3-26,23-
lactone exhibited approximately 850-fold stronger antagonistic
activity (IC50 ) 7.4 pM) than 2 (IC50 ) 6.3 nM).
We expected that the analogs with potent anti-D activity
would contribute to understanding the mechanisms involved in
the expression of antagonistic activity on VDR, for example,
based on X-ray crystallographic data if available,37 as well as
to finding the seeds of new medicines for treating Paget’s
disease.
229
388
291
3444
a The potency of the natural hormone 1 is normalized to 100. b The
potency of 2 (IC50 ) 6.0∼11.0 nM) is normalized to 100 (see Experimental
Section). c See ref 24a.
Table 5. Biological Activities of 2R-Modified (23R)-24-alkylvitamin D3
Lactones (82a-91a, 82b-91b, and 82c-91c)
VDR
relative
antagonistic
activityb
VDR
relative
antagonistic
activityb
binding
binding
compd
affinitya
compd
affinitya
3ac
3bc
3cc
37.0
33.3
22.7
38
100
66
82a
82b
82c
83a
83b
83c
84a
84b
84c
85a
85b
85c
86a
86b
86c
11.9
38.5
33.3
6.54
15.4
7.04
4.39
10.6
6.41
7.46
15.4
8.00
0.97
5.24
3.79
85
8
87a
87b
87c
88a
88b
88c
89a
89b
89c
90a
90b
90c
91a
91b
91c
4.90
33.3
13.2
7.09
20.8
7.46
8.77
30.3
13.5
5.18
16.7
11.6
1.52
14.1
6.13
476
30
41
74
18
12
18
37
25
12
573
124
1
303
88
4
32
42
Experimental Section
General Procedure for the Synthesis of Vitamin D3 Lac-
tones: Method A. To a solution of an A-ring precursor and the
CD-ring precursor in toluene were added Et3N and Pd(PPh3)4 (30
mol % to the CD-ring precursor), and the mixture was stirred at
110 °C. After the mixture was concentrated, the residue was roughly
purified by flash column chromatography on silica gel to give a
crude vitamin D3. The crude product was dissolved in MeOH. To
the MeOH solution was added (+)-10-camphorsulfonic acid (CSA)
at 0 °C, and the mixture was stirred at room temperature. To the
mixture was added saturated NaHCO3 aq solution, and the aqueous
layer was extracted with AcOEt. The organic layer was washed
with saturated NaCl aq solution, dried over Na2SO4, and concen-
trated. The residue was purified by flash column chromatography
on silica gel or preparative thin-layer chromatography on silica gel
to give the vitamin D3 lactone derivative. Further purification for
biological assays was conducted by reverse-phase recycle HPLC
(YMC-PacK ODS column, 20 × 150 mm, 9.9 mL/min, CH3CN/
H2O ) 95:5-85:15).
General Procedure for the Synthesis of Vitamin D3 Lac-
tones: Method B. To a solution of an A-ring precursor (1.5 equiv
to a CD-ring precursor) and the CD-ring precursor in toluene were
added Et3N and Pd(PPh3)4 (30 mol % to the CD-ring precursor),
and the mixture was stirred at 110 °C. After the mixture was
concentrated, the residue was roughly purified by flash column
chromatography on silica gel to give a crude vitamin D3. The crude
product was dissolved in MeCN. To the solution was added a 10%
solution of HF (46% aqueous solution, commercially available) in
MeCN at 0 °C, and the mixture was stirred at room temperature.
To the mixture was added saturated NaHCO3 aq solution, and the
aqueous layer was extracted with AcOEt. The organic layer was
washed with saturated NaCl aq solution, dried over Na2SO4, and
concentrated. The residue was purified by flash column chroma-
tography on silica gel or preparative thin-layer chromatography on
27
170
117
97
102
46
37
50
31
25
a The potency of the natural hormone 1 is normalized to 100. b The
potency of 2 (IC50 ) 6.0∼11.0 nM) is normalized to 100 (see Experimental
Section). c See ref 24a.
the R-methylene-γ-lactone of 2 and its analogs Via 1,4-addition
to give the corresponding cysteine adduct.36 Such interaction
between the ligand and the LBD might prevent correct position-
ing of helix 12 to activate the target genes. Therefore, it is
conceivable that the vitamin D antagonists, whose exo-meth-
ylene moiety is located at a more favorable position to react
with Cys403 and/or Cys410 after binding, show stronger vitamin
D antagonistic activity. The novel vitamin D3 lactones we
synthesized, which showed more potent antagonistic activity,
might be situated in a preferable position of the exo-methylene
group toward the cysteine residues after binding to the LBD of
the VDR and vice versa. This hypothesis on mechanism of
antagonism, right positioning of functional groups in the LBD
of the VDR, is different from that of 92 (ZK-159222)19 and 93
(DLAMs)20b (Figure 6), and would be one reason why the
potency of antagonistic activity of 2 and its analogs does not
correlate to the VDR binding affinity.

7070 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Saito et al.
silica gel to give the vitamin D3 lactone derivative. Further
purification for biological assays was conducted by reversed-phase
recycle HPLC (YMC-Pack ODS column, 20 × 150 mm, 9.9 mL/
min, CH3CN/H2O ) 95:5-85:15).
(23S,24S)-25-Dehydro-24-butyl-1R-hydroxyvitamin D3-26,23-
lactone (75). According to the general procedure (method B), a
crude product, which was obtained from 25 (40.0 mg, 94.5 µmol),
14 (52.2 mg, 0.142 mol), Et3N (3 mL), and Pd(PPh3)4 (32.8 mg,
28.4 µmol) in toluene (3 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 75
(23S,24S)-25-Dehydro-1R-hydroxy-24-methylvitamin D3-26,-
23-lactone (72). According to the general procedure (method A),
a crude product, which was obtained from 22 (34.9 mg, 91.5 µmol),
14 (44.0 mg, 0.119 mmol), Et3N (1.5 mL), and Pd(PPh3)4 (31.9
mg, 27.6 µmol) in toluene (3 mL) at 110 °C for 1.5 h, was treated
with CSA (42.1 mg, 0.181 mmol) in MeOH (2 mL) for 1.5 h. After
the usual work up, the crude product was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 1/3) to give 72
(19.7 mg, 48% in 2 steps) as a colorless oil. [R]18D -17.0 (c 0.52,
(28.0 mg, 61% in 2 steps) as a colorless amorphous solid. [R]22
D
-13.3 (c 1.07, CHCl3); IR (film, CHCl3) 3368, 1759, 1663, 1647,
1
1348, 1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H),
0.92 (t, J ) 7.1 Hz, 3H), 1.06 (d, J ) 6.3 Hz, 3H), 1.20-1.78 (m,
19H), 1.88-2.08 (m, 5H), 2.32 (dd, J ) 13.5, 6.4 Hz, 1H), 2.60
(dd, J ) 13.5, 3.4 Hz, 1H), 2.83 (m, 1H), 2.89 (m, 1H), 4.23 (m,
1H), 4.43 (m, 1H), 4.57 (ddd, J ) 8.5, 6.3, 5.1 Hz, 1H), 5.00 (s,
1H), 5.33 (s, 1H), 5.50 (d, J ) 2.0 Hz, 1H), 6.02 (d, J ) 11.2 Hz,
1H), 6.19 (d, J ) 2.0 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C
NMR (150 MHz, CDCl3) δ 11.9, 13.9, 19.7, 22.2, 22.6, 23.5, 26.8,
27.8, 28.4, 29.0, 34.5, 35.9, 40.3, 42.8, 43.6, 45.2, 45.9, 56.1, 56.8,
66.8, 70.8, 80.6, 111.7, 117.2, 120.9, 124.8, 133.1, 139.7, 142.7,
147.6, 170.7; EI-LRMS m/z 482 (M+), 464, 446, 251, 153;
EI-HRMS calcd for C31H46O4, 482.3396; found, 482.3487.
1
CHCl3); IR (neat) 3395, 1755, 1638, 1269, 1055 cm-1; H NMR
(400 MHz, CDCl3) δ 0.56 (s, 3H), 1.05 (d, J ) 6.6 Hz, 3H), 1.13
(d, J ) 7.1 Hz, 3H), 1.20-1.75 (m, 13H), 1.87-1.95 (m, 2H),
1.96-2.08 (m, 3H), 2.31 (dd, J ) 13.4, 6.6 Hz, 1H), 2.59 (dd, J )
13.4, 3.4 Hz, 1H), 2.82 (dd, J ) 12.5, 4.4 Hz, 1H), 3.11 (dddq, J
) 2.2, 2.2, 6.8, 7.1 Hz, 1H), 4.22 (m, 1H), 4.43 (m, 1H), 4.59
(ddd, J ) 8.2, 6.8, 5.3 Hz, 1H), 4.99 (dd, J ) 1.5, 1.5 Hz, 1H),
5.32 (dd, J ) 1.5, 1.5 Hz, 1H), 5.53 (d, J ) 2.2 Hz, 1H), 6.01 (d,
J ) 11.2 Hz, 1H), 6.18 (d, J ) 2.2 Hz, 1H), 6.37 (d, J ) 11.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 12.1, 14.7, 19.9, 22.4, 23.6,
27.8, 29.1, 34.5, 36.4, 38.2, 40.8, 42.9, 45.3, 46.0, 56.2, 56.9, 66.8,
70.8, 80.3, 111.6, 117.1, 120.4, 124.3, 132.9, 141.3, 142.6, 147.5,
170.2; EI-LRMS m/z 440 (M+), 422, 404, 251, 105; EI-HRMS calcd
for C28H40O4, 440.2987; found, 440.2932.
(23S,24S)-25-Dehydro-1R-hydroxy-24-isobutylvitamin D3-26,-
23-lactone (76). According to the general procedure (method B),
a crude product, which was obtained from 26 (15.7 mg, 37.1 µmol),
14 (20.5 mg, 55.6 µmol), Et3N (2 mL), and Pd(PPh3)4 (12.9 mg,
11.2 µmol) in toluene (2 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 76
(23S,24S)-25-Dehydro-24-ethyl-1R-hydroxyvitamin D3-26,23-
lactone (73). According to the general procedure (method B), a
crude product, which was obtained from 23 (19 mg, 49 µmol), 14
(24.0 mg, 65.1 µmol), Et3N (3.0 mL), and Pd(PPh3)4 (15.0 mg,
12.9 µmol) in toluene (0.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 3 h. After the usual
work up, the crude product was purified by flash column chroma-
tography on silica gel (hexane/AcOEt ) 1/1) to give 73 (13.0 mg,
(8.3 mg, 46% in 2 steps) as a colorless amorphous solid. [R]26
D
-11.4 (c 0.64, CHCl3); IR (neat) 3384, 1763, 1663, 1644, 1346,
1
1269, 1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H),
0.95 (d, J ) 6.3 Hz, 6H), 1.06 (d, J ) 6.4 Hz, 3H), 1.20-1.75 (m,
16H), 1.85-2.10 (m, 5H), 2.32 (dd, J ) 13.4, 6.6 Hz, 1H), 2.60
(dd, J ) 13.4, 2.8 Hz, 1H), 2.83 (m, 1H), 3.02 (m, 1H), 4.23 (m,
1H), 4.43 (m, 1H), 4.58 (m, 1H), 5.00 (s, 1H), 5.33 (s, 1H), 5.48
(d, J ) 1.7 Hz, 1H), 6.02 (d, J ) 11.2 Hz, 1H), 6.19 (d, J ) 1.7
Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C NMR (150 MHz, CDCl3)
δ 11.9, 19.8, 21.9, 22.3, 23.1, 24.4, 27.8, 29.0, 34.6, 36.0, 36.1,
40.4, 41.5, 42.9, 45.2, 46.0, 46.1, 56.1, 56.8, 66.9, 70.8, 80.6, 111.8,
117.1, 120.6, 124.9, 133.0, 140.0, 142.8, 147.6, 170.7; EI-LRMS
m/z 482 (M+), 464, 446, 251, 153; EI-HRMS calcd for C31H46O4,
482.3396; found, 482.3401.
67% in 2 steps) as a colorless oil. [R]23 -20.6 (c 1.00, CHCl3);
D
IR (neat) 3366, 2924, 1761, 1653, 1061 cm-1; 1H NMR (400 MHz,
CDCl3) δ 0.56 (s, 3H), 0.95 (t, J ) 7.4 Hz, 3H), 1.05 (d, J ) 6.3
Hz, 3H), 1.25-1.77 (m, 15H), 1.88-2.05 (m, 5H), 2.31 (dd, J )
13.3, 6.6 Hz, 1H), 2.59 (dd, J ) 13.3, 3.3 Hz, 1H), 2.78-2.84 (m,
2H), 4.23 (m, 1H), 4.43 (dd, J ) 7.7, 4.3 Hz, 1H), 4.58 (m, 1H),
4.99 (s, 1H), 5.32 (s, 1H), 5.51 (d, J ) 1.8 Hz, 1H), 6.01 (d, J )
11.2 Hz, 1H), 6.20 (d, J ) 1.8 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 10.9, 12.0, 19.8, 20.2, 22.3, 23.6,
27.8, 29.1, 34.6, 35.9, 40.4, 42.9, 45.2, 45.3, 46.0, 56.2, 56.9, 66.8,
70.8, 80.5, 111.7, 117.1, 121.1, 124.8, 133.0, 139.4, 142.7, 147.6,
170.6; EI-LRMS m/z 454 (M+), 436, 418, 322, 249; EI-HRMS calcd
for C29H42O4, 454.3083; found, 454.3088.
(23S,24R)-25-Dehydro-1R-hydroxy-24-methylvitamin D3-26,-
23-lactone (77). According to the general procedure (method A),
a crude product, which was obtained from 67 (14.0 mg, 36.7 µmol),
14 (17.8 mg, 48.3 µmol), Et3N (1 mL), and Pd(PPh3)4 (13.0 mg,
11.2 µmol) in toluene (2 mL) at 110 °C for 1.5 h, was treated with
CSA (20.0 mg, 86.1 µmol) in MeOH (1.5 mL) for 1.5 h. After the
usual work up, the crude product was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 1/2) to give 77
(23S,24S)-25-Dehydro-1R-hydroxy-24-propylvitamin D3-26,-
23-lactone (74). According to the general procedure (method B),
a crude product, which was obtained from 24 (26.9 mg, 65.7 µmol),
14 (36.3 mg, 98.5 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (22.8 mg,
19.7 µmol) in toluene (1.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (4 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 74
(7.8 mg, 48% in 2 steps) as an amorphous solid. [R]23 +19.7 (c
D
1
0.30, CHCl3); IR (neat) 3400, 1759, 1630 cm-1; H NMR (400
MHz, CDCl3) δ 0.57 (s, 3H), 1.06 (d, J ) 5.9 Hz, 3H), 1.28 (d, J
) 6.8 Hz, 3H), 1.25-1.80 (m, 13H), 1.85-2.10 (m, 5H), 2.32 (dd,
J ) 13.6, 6.4 Hz, 1H), 2.55-2.70 (m, 2H), 2.83 (m, 1H), 4.07 (dt,
J ) 5.9, 6.4 Hz, 1H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (s, 1H),
5.33 (s, 1H), 5.53 (d, J ) 2.9 Hz, 1H), 6.01 (d, J ) 11.1 Hz, 1H),
6.22 (d, J ) 2.9 Hz, 1H), 6.37 (d, J ) 11.1 Hz, 1H); 13C NMR
(150 MHz, CDCl3) δ 12.0, 17.2, 19.5, 22.3, 23.5, 27.9, 29.0, 34.5,
40.4, 40.7, 41.5, 42.9, 45.2, 45.9, 56.2, 56.5, 66.9, 70.8, 84.2, 111.8,
117.2, 120.8, 124.9, 133.0, 140.8, 142.8, 147.6, 170.4; EI-LRMS
m/z 440 (M+), 422, 404, 251, 105; EI-HRMS calcd for C28H40O4,
440.2927; found, 440.2929.
(23S,24R)-25-Dehydro-24-ethyl-1R-hydroxyvitamin D3-26,23-
lactone (78). According to the general procedure (method B), a
crude product, which was obtained from 68 (14.0 mg, 35.4 µmol),
14 (20.0 mg, 54.2 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (12.0 mg,
10.4 µmol) in toluene (0.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 3 h. After the usual
(15.8 mg, 51% in 2 steps) as a colorless amorphous solid. [R]18
D
-14.8 (c 1.22, CHCl3); IR (film, CHCl3) 3376, 1759, 1661, 1643,
1
1348, 1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H),
0.95 (t, J ) 7.0 Hz, 3H), 1.06 (d, J ) 6.6 Hz, 3H), 1.20-1.80 (m,
17H), 1.88-2.08 (m, 5H), 2.32 (dd, J ) 13.4, 6.6 Hz, 1H), 2.60
(dd, J ) 13.4, 3.4 Hz, 1H), 2.83 (m, 1H), 2.91 (m, 1H), 4.23 (m,
1H), 4.43 (m, 1H), 4.58 (ddd, J ) 8.6, 6.2, 4.9 Hz, 1H), 5.00 (s,
1H), 5.33 (s, 1H), 5.50 (d, J ) 1.8 Hz, 1H), 6.02 (d, J ) 11.2 Hz,
1H), 6.19 (d, J ) 1.8 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C
NMR (150 MHz, CDCl3) δ 11.9, 14.0, 19.4, 19.8, 22.3, 23.5,
27.8, 29.0, 29.2, 34.6, 36.0, 40.4, 42.9, 43.4, 45.2, 46.0, 56.1, 56.8,
66.8, 70.8, 80.6, 111.7, 117.2, 120.9, 124.9, 133.0, 139.7, 142.8,
147.6, 170.7; EI-LRMS m/z 468 (M+), 450, 432, 417, 263, 251,
209, 195, 155, 141; EI-HRMS calcd for C30H44O4, 468.3240; found,
468.3233.

Synthetic and Biological Studies on Vitamin D Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7071
work up, the crude product was purified by flash column chroma-
(m, 1H), 4.15-4.25 (m, 2H), 4.43 (m, 1H), 4.99 (s, 1H), 5.32 (s,
1H), 5.57 (d, J ) 2.3 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H), 6.24 (d,
J ) 2.3 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C NMR (150 MHz,
CDCl3) δ 11.9, 19.6, 22.2, 22.3, 22.7, 23.5, 25.1, 27.9, 29.0, 34.3,
40.3, 42.3, 42.9, 43.0, 44.0, 45.2, 45.9, 56.2, 56.5, 66.8, 70.8, 83.0,
111.7, 117.2, 122.1, 124.9, 133.1, 139.7, 142.8, 147.6, 170.5;
EI-LRMS m/z 482 (M+), 464, 446, 251, 153; EI-HRMS calcd for
C31H46O4, 482.3396; found, 482.3397.
tography on silica gel (hexane/AcOEt ) 2/3) to give 78 (9.0 mg,
56% in 2 steps) as a colorless oil. [R]22 +34.8 (c 0.69, CHCl3);
D
IR (neat) 3441, 2936, 1757, 1649, 1059 cm-1; 1H NMR (400 MHz,
CDCl3) δ 0.56 (s, 3H), 0.97 (t, J ) 7.4 Hz, 3H), 1.05 (d, J ) 5.9
Hz, 3H), 1.18-1.69 (m, 16H), 1.86-2.05 (m, 4H), 2.31 (dd, J )
13.3, 6.5 Hz, 1H), 2.54-2.61 (m, 2H), 2.82 (m, 1H), 4.24 (m, 2H),
4.43 (m, 1H), 4.99 (s, 1H), 5.32 (s, 1H), 5.59 (d, J ) 2.3 Hz, 1H),
6.01 (d, J ) 11.2 Hz, 1H), 6.27 (d, J ) 2.3 Hz, 1H), 6.37 (d, J )
11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 10.7, 12.0, 19.7,
22.3, 23.6, 26.8, 28.0, 29.1, 32.2, 40.4, 42.5, 42.9, 45.3, 45.9, 46.4,
56.2, 56.5, 66.8, 70.8, 82.1, 111.7, 117.1, 122.2, 124.8, 133.0, 138.8,
142.7, 147.8, 170.4; EI-LRMS m/z 454 (M+), 436, 418, 322, 249;
EI-HRMS calcd for C29H42O4, 454.3083; found, 454.3086.
(23R,24S)-25-Dehydro-1R-hydroxy-24-methylvitamin D3-26,-
23-lactone (82). According to the general procedure (method A),
a crude product, which was obtained from 57 (18.8 mg, 49.3 µmol),
14 (27.3 mg, 74.0 µmol), Et3N (1 mL), and Pd(PPh3)4 (17.1 mg,
14.8 µmol) in toluene (2 mL) at 110 °C for 1.5 h, was treated with
CSA (27.0 mg, 0.116 mmol) in MeOH (1.5 mL) for 1.5 h. After
the usual work up, the crude product was purified by flash column
chromatography on silica gel (hexane/AcOEt ) 1/2) to give 82
(23S,24R)-25-Dehydro-1R-hydroxy-24-propylvitamin D3-26,-
23-lactone (79). According to the general procedure (method B),
a crude product, which was obtained from 69 (36.0 mg, 87.9 µmol),
14 (48.6 mg, 0.132 mmol), Et3N (1.5 mL), and Pd(PPh3)4 (30.5
mg, 26.4 µmol) in toluene (1.5 mL) at 110 °C for 1 h, was treated
with a 5% solution of HF in MeCN (4 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 79
(7.8 mg, 52% in 2 steps) as an amorphous solid. [R]18 +81.3 (c
D
1
0.27, CHCl3); IR (neat) 3383, 1765, 1643, 1247, 1057 cm-1; H
NMR (400 MHz, CDCl3) δ 0.57 (s, 3H), 1.01 (d, J ) 6.4 Hz, 3H),
1.22 (d, J ) 6.8 Hz, 3H), 1.20-1.38 (m, 4H), 1.40-2.10 (m, 14H),
2.31 (dd, J ) 13.4, 6.4 Hz, 1H), 2.55-2.65 (m, 2H), 2.82 (dd, J )
12.2, 3.9 Hz, 1H), 4.07 (ddd, J ) 10.5, 7.3, 2.0 Hz, 1H), 4.22 (m,
1H), 4.42 (dd, J ) 7.6, 4.4 Hz, 1H), 4.99 (s, 1H), 5.32 (dd, J )
1.7, 1.4 Hz, 1H), 5.52 (d, J ) 2.9 Hz, 1H), 6.01 (d, J ) 11.2 Hz,
1H), 6.21 (d, J ) 2.9 Hz, 1H), 6.36 (d, J ) 11.2 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 12.2, 16.3, 18.8, 22.4, 23.6, 27.7, 29.1,
33.1, 40.6, 40.9, 41.6, 43.0, 45.3, 46.1, 56.4, 56.9, 66.9, 70.8, 82.5,
111.7, 117.1, 120.4, 124.7, 133.0, 140.8, 142.5, 147.4, 170.2;
EI-LRMS m/z 440 (M+), 422, 404, 251, 105; EI-HRMS calcd for
C28H44O4, 440.2927; found, 440.2920
(20.5 mg, 50% in 2 steps) as a colorless amorphous solid. [R]15
D
+32.7 (c 1.15, CHCl3); IR (film, CHCl3) 3387, 1761, 1661, 1647,
1
1601, 1348, 1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s,
3H), 0.96 (t, J ) 7.2 Hz, 3H), 1.06 (d, J ) 5.9 Hz, 3H), 1.15-1.73
(m, 17H), 1.85-2.08 (m, 5H), 2.32 (dd, J ) 13.4, 6.4 Hz, 1H),
1.56-2.65 (m, 2H), 2.82 (m, 1H), 4.18-4.30 (m, 2H), 4.43 (m,
1H), 4.99 (s, 1H), 5.32 (dd, J ) 1.7, 1.5 Hz, 1H), 5.58 (d, J ) 2.3
Hz, 1H), 6.01 (d, J ) 11.4 Hz, 1H), 6.25 (d, J ) 2.3 Hz, 1H), 6.37
(d, J ) 11.4 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 11.9, 14.0,
19.5, 19.6, 22.2, 23.5, 27.9, 29.0, 34.2, 36.4, 40.3, 42.4, 42.9, 44.9,
45.2, 45.9, 56.2, 56.5, 66.8, 70.7, 82.5, 111.7, 117.2, 122.2, 124.9,
133.1, 139.2, 142.7, 147.6, 170.5; EI-LRMS m/z 468 (M+), 450,
432, 417, 263, 251, 209, 195, 155, 141; EI-HRMS calcd for
C30H44O4, 468.3240; found, 468.3222.
(23R,24S)-25-Dehydro-24-ethyl-1R-hydroxyvitamin D3-26,23-
lactone (83). According to the general procedure (method B), a
crude product, which was obtained from 58 (14.0 mg, 35.4 µmol),
14 (20.0 mg, 54.2 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (12.0 mg,
10.4 µmol) in toluene (0.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 3 h. After the usual
work up, the crude product was purified by flash column chroma-
tography on silica gel (hexane/AcOEt ) 2/3) to give 83 (9.0 mg,
(23S,24R)-25-Dehydro-24-butyl-1R-hydroxyvitamin D3-26,23-
lactone (80). According to the general procedure (method B), a
crude product, which was obtained from 70 (34.7 mg, 81.9 µmol),
14 (45.3 mg, 0.123 mol), Et3N (3 mL), and Pd(PPh3)4 (28.4 mg,
24.6 µmol) in toluene (3.0 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 3 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 80
56% in 2 steps) as a colorless oil. [R]24 +50.7 (c 0.38, CHCl3);
D
IR (neat) 3366, 2946, 1751, 1630, 1061 cm-1; 1H NMR (400 MHz,
CDCl3) δ 0.56 (s, 3H), 0.98 (t, J ) 7.4 Hz, 3H), 1.02 (d, J ) 6.6
Hz, 3H), 1.23-1.85 (m, 16H), 1.86-2.05 (m, 4H), 2.31 (dd, J )
13.3, 6.5 Hz, 1H), 2.51 (m, 1H), 2.60 (dd, J ) 13.5, 3.4 Hz, 1H),
2.83 (dd, J ) 12.2, 3.9 Hz, 1H), 4.23 (m, 1H), 4.29 (ddd, J )
11.0, 4.9, 2.0 Hz, 1H), 4.43 (m, 1H), 5.00 (s, 1H), 5.33 (s, 1H),
5.5 (d, J ) 2.6 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H), 6.27 (d, J )
2.6 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 10.9, 12.1, 18.6, 22.3, 23.6, 26.4, 27.6, 29.1, 33.1, 40.6,
42.9, 43.3, 45.3, 46.0, 46.6, 56.4, 56.9, 66.9, 70.8, 80.6, 111.7,
117.2, 122.0, 124.8, 133.0, 139.1, 142.7, 147.5, 170.4; EI-LRMS
m/z 454 (M+), 436, 418, 322, 249; EI-HRMS calcd for C29H42O4,
454.3083; found, 454.3080.
(20.5 mg, 52% in 2 steps) as a colorless amorphous solid. [R]20
D
+29.2 (c 1.62, CHCl3); IR (film, CHCl3) 3403, 1759, 1647, 1663,
1
1348, 1053 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H),
0.92 (t, J ) 6.6 Hz, 3H), 1.06 (d, J ) 5.9 Hz, 3H), 1.15-1.75 (m,
19H), 1.85-2.10 (m, 5H), 2.32 (dd, J ) 13.3, 6.5 Hz, 1H), 2.55-
2.65 (m, 2H), 2.83 (m, 1H), 4.18-4.30 (m, 2H), 4.43 (m, 1H),
4.99 (s, 1H), 5.32 (s, 1H), 5.58 (d, J ) 2.1 Hz, 1H), 6.01 (d, J )
11.2 Hz, 1H), 6.23 (d, J ) 2.1 Hz, 1H), 6.26 (d, J ) 11.2 Hz, 1H);
13C NMR (150 MHz, CDCl3) δ 11.9, 13.9, 10.6, 22.2, 22.6, 23.5,
27.9, 28.4, 29.0, 33.8, 34.2, 40.3, 42.4, 42.8, 45.0, 45.2, 45.9, 56.2,
56.5, 66.8, 70.7, 82.5, 111.7, 117.2, 122.2, 124.9, 133.1, 139.2,
142.7, 147.6, 170.5; EI-LRMS m/z 482 (M+), 464, 446, 251, 153;
EI-HRMS calcd for C31H46O4, 482.3396; found, 482.3399.
(23R,24S)-25-Dehydro-1R-hydroxy-24-propylvitamin D3-26,-
23-lactone (84). According to the general procedure (method B),
a crude product, which was obtained from 59 (21.3 mg, 52.0 µmol),
14 (28.8 mg, 78.1 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (18.0 mg,
15.6 µmol) in toluene (1.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (4 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 84
(23S,24R)-25-Dehydro-1R-hydroxy-24-isobutylvitamin D3-26,-
23-lactone (81). According to the general procedure (method B),
a crude product, which was obtained from 71 (21.2 mg, 50.0 µmol),
14 (27.7 mg, 75.1 µmol), Et3N (2 mL), and Pd(PPh3)4 (17.3 mg,
15.0 µmol) in toluene (2 mL) at 110 °C for 1.5 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 81
(10.3 mg, 42% in 2 steps) as a colorless amorphous solid. [R]15
D
+57.0 (c 0.623, CHCl3); IR (film, CHCl3) 3387, 1759, 1661, 1644,
1634, 1609, 1350, 1053 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.57
(s, 3H), 0.96 (t, J ) 7.3 Hz, 3H), 1.02 (d, J ) 6.6 Hz, 3H), 1.20-
2.10 (m, 22H), 2.31 (dd, J ) 13.4, 6.4 Hz, 1H), 2.53-2.63 (m,
2H), 2.83 (m, 1H), 4.20-4.35 (m, 2H), 4.43 (m, 1H), 5.00 (s, 1H),
5.33 (s, 1H), 5.57 (d, J ) 2.6 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H),
6.25 (d, J ) 2.6 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C NMR
(150 MHz, CDCl3) δ 12.0, 14.1, 18.5, 19.8, 22.3, 23.5, 27.6, 29.0,
33.0, 35.9, 40.5, 42.8, 43.2, 45.1, 45.2, 46.0, 56.3, 56.9, 66.8, 70.8,
81.0, 111.8, 117.2, 121.9, 124.9, 133.1, 139.6, 142.8, 147.6, 170.5;
(10.3 mg, 43% in 2 steps) as a colorless amorphous solid. [R]26
D
+28.0 (c 0.79, CHCl3); IR (neat) 3389, 1761, 1657, 1269, 1140,
1
1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H), 0.96 (d,
J ) 6.6 Hz, 3H), 0.97 (d, J ) 6.6 Hz, 3H), 1.06 (d, J ) 6.1 Hz,
3H), 1.10-1.75 (m, 16H), 1.85-2.10 (m, 5H), 2.32 (dd, J ) 13.4,
6.6 Hz, 1H), 2.60 (dd, J ) 13.4, 3.6 Hz, 1H), 2.67 (m, 1H), 2.83

7072 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Saito et al.
EI-LRMS m/z 468 (M+), 450, 432, 417, 263, 251, 209, 195, 155,
141; EI-HRMS calcd for C30H44O4, 468.3239; found, 468.3228.
(23R,24S)-25-Dehydro-24-butyl-1R-hydroxyvitamin D3-26,23-
lactone (85). According to the general procedure (method B), a
crude product, which was obtained from 60 (40.0 mg, 94.5 µmol),
14 (52.2 mg, 0.145 mol), Et3N (3 mL), and Pd(PPh3)4 (28.4 mg,
24.6 µmol) in toluene (3.0 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 85
14 (25.0 mg, 67.9 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (16.0 mg,
13.8 µmol) in toluene (0.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 3 h. After the usual
work up, the crude product was purified by flash column chroma-
tography on silica gel (hexane/AcOEt ) 1/1) to give 88 (13.0 mg,
63% in 2 steps) as a colorless oil. [R]22 +75.7 (c 0.92, CHCl3);
D
IR (neat) 3416, 2934, 1755, 1649, 1055 cm-1; 1H NMR (400 MHz,
CDCl3) δ 0.56 (s, 3H), 0.98 (t, J ) 7.3 Hz, 3H), 1.05 (d, J ) 6.6
Hz, 3H), 1.12 (ddd, J ) 14.0, 10.9, 1.8 Hz, 1H), 1.25-2.04 (m,
19H), 2.31 (dd, J ) 13.5, 6.3 Hz, 1H), 2.59 (dd, J ) 13.5, 2.8 Hz,
1H), 2.80-2.90 (m, 2H), 4.23 (m, 1H), 4.43 (J ) 7.8, 4.2 Hz, 1H),
4.66 (ddd, J ) 11.5, 7.0, 1.5 Hz, 1H), 4.99 (s, 1H), 5.33 (s, 1H),
5.52 (d, J ) 2.6 Hz, 1H), 6.01 (d, J ) 11.4 Hz, 1H), 6.21 (d, J )
2.6 Hz, 1H), 6.36 (d, J ) 11.4 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 11.3, 12.1, 18.6, 20.5, 22.3, 23.6, 27.6, 29.1, 32.7, 36.4,
40.6, 42.9, 45.1, 45.3, 46.0, 56.4, 57.1, 66.8, 70.8, 78.3, 111.7,
117.2, 120.9, 124.8, 133.0, 139.3, 142.6, 147.5, 170.6; EI-LRMS
m/z 454 (M+), 436, 418, 322, 249; EI-HRMS calcd for C29H42O4,
454.3083; found, 454.3083.
(22.9 mg, 52% in 2 steps) as a colorless amorphous solid. [R]20
D
+57.2 (c 1.08, CHCl3); IR (film, CHCl3) 3387, 1759, 1659, 1647,
1
1269, 1053 cm-1; H NMR (400 MHz, CDCl3) δ 0.57 (s, 3H),
0.93 (t, J ) 7.0 Hz, 3H), 1.03 (d, J ) 6.6 Hz, 3H), 1.20-2.08 (m,
24H), 2.31 (dd, J ) 13.6, 6.4 Hz, 1H), 2.55 (m, 1H), 2.60 (dd, J
) 13.6, 3.1 Hz, 1H), 2.83 (m, 1H), 4.23 (m, 1H), 4.27 (ddd, J )
11.1, 4.9, 2.1 Hz, 1H), 4.43 (m, 1H), 5.00 (s, 1H), 5.33 (s, 1H),
5.57 (d, J ) 2.6 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H), 6.26 (d, J )
2.6 Hz, 1H), 6.37 (d, J ) 11.2 Hz, 1H); 13C NMR (150 MHz,
CDCl3) δ 12.0, 13.9, 18.5, 22.2, 22.7 (2C), 23.5, 27.6, 28.6, 29.0,
33.0, 33.4, 40.5, 42.8, 43.1, 45.2, 46.0, 56.3, 56.9, 66.8, 70.8, 81.0,
111.8, 117.2, 121.9, 124.8, 133.1, 139.6, 142.7, 147.6, 170.5;
EI-LRMS m/z 482 (M+), 464, 446, 251, 153; EI-HRMS calcd for
C31H46O4, 482.3396; found, 482.3398.
(23R,24R)-25-Dehydro-1R-hydroxy-24-propylvitamin D3-26,-
23-lactone (89). According to the general procedure (method B),
a crude product, which was obtained from 29 (25.5 mg, 62.3 µmol),
14 (34.5 mg, 93.6 µmol), Et3N (1.5 mL), and Pd(PPh3)4 (21.6 mg,
18.7 µmol) in toluene (1.5 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (4 mL) for 3 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 89
(23R,24S)-25-Dehydro-1R-hydroxy-24-isobutylvitamin D3-26,-
23-lactone (86). According to the general procedure (method B),
a crude product, which was obtained from 61 (20.4 mg, 48.2 µmol),
14 (26.6 mg, 72.1 µmol), Et3N (2 mL), and Pd(PPh3)4 (16.7 mg,
14.5 µmol) in toluene (2 mL) at 110 °C for 1 h, was treated with
a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 86
(13.0 mg, 52% in 2 steps) as a colorless amorphous solid. [R]18
D
+108.6 (c 1.16, CHCl3); IR (film, CHCl3) 3382, 1757, 1663, 1645,
1
1346, 1055 cm-1; H NMR (400 MHz, CDCl3) δ 0.57 (s, 3H),
0.96 (t, J ) 7.1 Hz, 3H), 1.00 (d, J ) 6.6 Hz, 3H), 1.11 (ddd, J )
14.0, 10.6, 1.8 Hz, 1H), 1.20-2.08 (m, 21H), 2.31 (dd, J ) 13.4,
6.4 Hz, 1H), 2.60 (dd, J ) 13.4, 3.3 Hz, 1H), 2.83 (m, 1H), 2.98
(m, 1H), 4.23 (m, 1H), 4.43 (m, 1H), 4.66 (ddd, J ) 11.6, 7.2, 1.6
Hz, 1H), 5.00 (s, 1H), 5.33 (s, 1H), 5.50 (d, J ) 2.4 Hz, 1H), 6.01
(d, J ) 11.2 Hz, 1H), 6.21 (d, J ) 2.4 Hz, 1H), 6.36 (d, J ) 11.2
Hz, 1H); 13C NMR (150 MHz, CDCl3) δ 12.0, 14.0, 18.5, 19.9,
22.2, 23.5, 27.6, 29.0, 29.5, 32.6, 36.5, 40.5, 42.8, 43.2, 45.2, 46.0,
56.4, 57.0, 66.8, 70.7, 78.3, 111.8, 117.2, 120.8, 124.8, 133.1, 139.5,
142.7, 147.6, 170.7; EI-LRMS m/z 468 (M+), 450, 432, 417, 263,
251, 209, 195, 155, 141; EI-HRMS calcd for C30H44O4, 468.3240;
found, 468.3231.
(14.4 mg, 62% in 2 steps) as a colorless amorphous solid. [R]24
D
+49.3 (c 1.11, CHCl3); IR (neat) 3385, 1761, 1659, 1381, 1143,
1
1057 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H), 0.95 (d,
J ) 6.4 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 3H), 1.02 (d, J ) 6.6 Hz,
3H), 1.20-1.35 (m, 4H), 1.40-2.08 (m, 17H), 2.31 (dd, J ) 13.3,
6.5 Hz, 1H), 2.60 (d, J ) 13.3, 2.9 Hz, 1H), 2.63 (m, 1H), 2.83
(m, 1H), 4.18-4.30 (m, 2H), 4.43 (m, 1H), 5.00 (s, 1H), 5.33 (s,
1H), 5.56 (d, J ) 2.3 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H), 6.24 (d,
J ) 2.3 Hz, 1H), 6.36 (d, J ) 11.2 Hz, 1H); 13C NMR (150 MHz,
CDCl3) δ 12.0, 18.5, 22.2, 22.3, 22.8, 23.5, 25.2, 27.6, 29.0, 33.0,
40.5, 42.8, 43.0, 43.1, 43.5, 45.2, 46.0, 56.3, 56.9, 66.8, 70.8, 81.4,
112.0, 117.2, 121.8, 124.8, 133.1, 140.0, 142.7, 147.6, 170.5;
EI-LRMS m/z 482 (M+), 464, 446, 251, 153; EI-HRMS calcd for
C31H46O4, 482.3396; found, 482.3797.
(23R,24R)-25-Dehydro-1R-hydroxy-24-methylvitamin D3-26,-
23-lactone (87). According to the general procedure (method A),
a crude product, which was obtained from 27 (36.5 mg, 95.7 µmol),
14 (45.9 mg, 0.124 mmol), Et3N (1.5 mL), and Pd(PPh3)4 (33.2
mg, 28.7 µmol) in toluene (3 mL) at 110 °C for 1.5 h, was treated
with CSA (48.8 mg, 0.201 mmol) in MeOH (1.5 mL) for 45 min.
After the usual work up, the crude product was purified by flash
column chromatography on silica gel (hexane/AcOEt ) 1/2) to give
87 (24.1 mg, 57% in 2 steps) as an amorphous solid. [R]23D +113.9
(c 0.38, CHCl3); IR (neat) 3420, 1757, 1658 cm-1; 1H NMR (400
MHz, CDCl3) δ 0.57 (s, 3H), 1.02 (d, J ) 6.4 Hz, 3H), 1.08 (m,
1H), 1.13 (d, J ) 7.3 Hz, 3H), 1.15-1.35 (m, 3H), 1.40-2.10 (m,
14H), 2.31 (dd, J ) 13.4, 6.6 Hz, 1H), 2.59 (dd, J ) 13.4, 3.3 Hz,
1H), 2.83 (dd, J ) 12.1, 3.8 Hz, 1H), 3.16 (dq, J ) 7.8, 7.3 Hz,
1H), 4.23 (m, 1H), 4.43 (m, 1H), 4.67 (ddd, J ) 11.8, 7.8, 2.0 Hz,
1H), 4.99 (s, 1H), 5.33 (s, 1H), 5.52 (d, J ) 2.7 Hz, 1H), 6.01 (d,
J ) 11.3 Hz, 1H), 6.21 (d, J ) 2.7 Hz, 1H), 6.36 (d, J ) 11.3 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 12.2, 13.9, 18.6, 22.4, 23.6,
27.7, 29.1, 32.6, 37.0, 37.8, 40.6, 42.9, 45.3, 46.1, 56.4, 57.1, 66.8,
70.7, 78.4, 111.7, 117.1, 120.5, 124.7, 133.0, 140.7, 142.5, 147.4,
170.2; EI-LRMS m/z 440 (M+), 422, 404, 378, 289, 209,105;
EI-HRMS calcd for C28H40O4, 440.2927; found, 440.2935.
(23R,24R)-25-Dehydro-24-ethyl-1R-hydroxyvitamin D3-26,23-
lactone (88). According to the general procedure (method B), a
crude product, which was obtained from 28 (18.0 mg, 45.7 µmol),
(23R,24R)-25-Dehydro-24-butyl-1R-hydroxyvitamin D3-26,-
23-lactone (90). According to the general procedure (method B),
a crude product, which was obtained from 30 (52.4 mg, 0.124
mmol), 14 (68.4 mg, 0.186 mmol), Et3N (3 mL), and Pd(PPh3)4
(43.0 mg, 37.2 µmol) in toluene (3.0 mL) at 110 °C for 1 h, was
treated with a 5% solution of HF in MeCN (2 mL) for 30 min.
After the usual work up, the crude product was purified by flash
column chromatography on silica gel (hexane/AcOEt ) 2/3) to give
90 (20.5 mg, 52% in 2 steps) as a colorless amorphous solid. [R]21
D
+58.4 (c 0.59, CHCl3); IR (film, CHCl3) 3351, 1758, 1658, 1643,
1379 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.57 (s, 3H), 0.93 (t, J
) 7.1 Hz, 3H), 1.01 (d, J ) 6.6 Hz, 3H), 1.11 (ddd, J ) 14.1,
10.8, 1.8 Hz, 1H), 1.20-2.08 (m, 23H), 2.31 (dd, J ) 13.4, 6.4 H,
1H), 2.59 (dd, J ) 13.4, 3.4 Hz, 1H), 2.83 (m, 1H), 2.96 (m, 1H),
4.23 (m, 1H), 4.43 (m, 1H), 4.66 (ddd, J ) 11.6, 7.1, 1.6 Hz, 1H),
4.99 (s, 1H), 5.33 (s, 1H), 5.51 (d, J ) 2.6 Hz, 1H), 6.01 (d, J )
11.2 Hz, 1H), 6.21 (d, J ) 2.6 Hz, 1H), 6.36 (d, J ) 11.2 Hz, 1H);
13C NMR (150 MHz, CDCl3) δ 12.0, 13.9, 18.5, 22.2, 22.6, 23.5.
27.0, 27.5, 28.8, 29.0, 32.6, 36.4, 40.5, 42.8, 43.4, 45.2, 46.0, 56.3,
57.0, 66.8, 70.7, 78.4, 111.8, 117.2, 120.8, 124.8, 133.2, 139.5,
142.7, 147.6, 170.7; EI-LRMS m/z 482 (M+), 464, 446, 251, 153;
EI-HRMS calcd for C31H46O4, 482.3397; found, 482.3387.
(23R,24R)-25-Dehydro-1R-hydroxy-24-isobutylvitamin D3-
26,23-lactone (91). According to the general procedure (method
B), a crude product, which was obtained from 31 (17.3 mg, 40.9
µmol), 14 (22.6 mg, 61.3 µmol), Et3N (2 mL), and Pd(PPh3)4 (14.2
mg, 12.3 µmol) in toluene (2 mL) at 110 °C for 1 h, was treated
with a 5% solution of HF in MeCN (2 mL) for 1 h. After the usual
work up, the crude product was purified by preparative thin-layer

Synthetic and Biological Studies on Vitamin D Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7073
chromatography on silica gel (hexane/AcOEt ) 2/3) to give 91
charts of assay for HL-60 cell differentiation to test antagonistic
activity are shown in the Supporting Information.
(12.5 mg, 63% in 2 steps) as a colorless amorphous solid. [R]25
D
+85.5 (c 0.95, CHCl3); IR (neat) 3378, 1759, 1662, 1346, 1269,
1
1053 cm-1; H NMR (400 MHz, CDCl3) δ 0.56 (s, 3H), 0.95 (d,
Acknowledgment. N.S. acknowledges the Takeda Science
Foundation for financial support. The authors thank Miss J.
Shimode, Miss M. Kitsukawa, and Miss A. Tonoki (Teikyo
University) for spectroscopic measurements. This study was
supported by Grants-in-aid from the Ministry of Education,
Culture, Sports, Science and Technology, Japan (N.S.) and Japan
Society for the Promotion of Science (A.K.). Uehara Memorial
Foundation is also acknowledged (A.K.). We are indebted to
Professor emeritus Hiroaki Takayama, Dr. Toshie Fujishima,
and Dr. Akihiro Yoshida for discussion on the early stage of
this work.
J ) 6.4 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 3H), 1.00 (d, J ) 6.4 Hz,
3H), 1.08 (m, 1H), 1.15-2.10 (m, 20H), 2.39 (dd, J ) 13.3, 6.5
Hz, 1H), 2.59 (dd, J ) 13.3, 2.9 Hz, 1H), 2.84 (m, 1H), 3.21 (m,
1H), 4.23 (br s, 1H), 4.43 (br s, 1H), 4.66 (m, 1H), 5.00 (s, 1H),
5.33 (s, 1H), 5.48 (d, J ) 2.2 Hz, 1H), 6.01 (d, J ) 11.2 Hz, 1H),
6.20 (d, J ) 2.2 Hz, 1H), 6.36 (d, J ) 11.2 Hz, 1H); 13C NMR
(150 MHz, CDCl3) δ 12.0, 18.5, 22.2, 22.5, 22.7, 23.5, 24.9, 27.6,
29.0, 32.6, 36.2, 36.7, 40.5, 41.2, 42.8, 45.2, 46.0, 56.4, 57.0, 66.8,
70.7, 78.3, 111.8, 117.2, 120.6, 124.8, 133.1, 139.6, 142.7, 147.6,
170.6; EI-LRMS m/z 482 (M+), 464, 446, 251, 153; EI-HRMS calcd
for C31H46O4, 482.3396; found, 482.3394.
Vitamin D Receptor (VDR) Binding Assay. [26,27-Methyl-
3H]-1R,25-dihydroxyvitamin D3 (specific activity 6.623 TBq/mmol,
15 000 dpm, 15.7 pg) and various amounts of 1R,25-dihydroxyvi-
tamin D3 and the analog to be tested were dissolved in 50 µL of
absolute ethanol in 12 × 75-mm polypropylene tubes. The chick
intestinal VDR (0.2 mg) and 1 mg of gelatin in 1 mL of phosphate
buffer solution (25 nM KH2PO4, 0.1 M KCl, and 1 mM dithio-
threitol, pH 7.4) were added to each tube in an ice bath. The assay
tubes were incubated in a shaking water bath for 1 h at 25 °C and
then chilled in an ice bath. One milliliter of 40% polypropylene
glycol 6000 in distilled water was added to each tube, which was
mixed vigorously and centrifuged at 2260 × g for 60 min at 4 °C.
After the supernatant was decanted, the bottom of the tube
containing the pellet was cut off into a scintillation vial con-
taining 10 mL of dioxane-based scintillation fluid, and the
radioactivity was measured with a Beckman liquid scintillation
counter (model LS6500). The relative potency of the analog was
calculated from the concentration needed to displace 50% of
[26,27-methyl-3H]-1R,25-dihydroxyvitamin D3 from the receptor
compared with the activity of 1R,25-dihydroxyvitamin D3 (assigned
a 100% value).
Assay for HL-60 Cell Differentiation. Nitro blue tetrazolium
(NBT)-reducing activity was used as a cell differentiation marker.
HL-60 cells were cultured in RPMI-1640 medium supplemented
with 10% heat-inactivated FCS. Exponentially proliferating cells
were collected, suspended in fresh medium, and seeded in culture
plates (Falcon, Becton Dickinson and Company, Franklin Lakes,
NJ). The cell concentration at seeding was adjusted to 2 × 104
cells/mL, and the seeding volume was 1 mL/well. An ethanol
solution of 1R,25-dihydroxyvitamin D3 (final concentration: 10-8
M) and an analog (final concentration: 3 × 10-12 to 10-6 M) was
added to the culture medium at 0.1% volume, and the culture was
continued for 96 h at 37 °C in a humidified atmosphere of 5%
CO2/air without a change of medium. The same amount of vehicle
was added to the control culture. The NBT-reducing assay was
performed according to the method of Collins.31 Briefly, cells were
collected, washed with PBS, and suspended in serum-free medium.
NBT/TPA solution (dissolved in PBS) was added. Final concentra-
tions of NBT and TPA were 0.1% and 100 ng/mL, respectively.
Then, the cell suspensions were incubated at 37 °C for 25 min.
After incubation, cells were collected by centrifugation and
resuspended in FCS. Cytospin smears were prepared, and the
counter-staining of nuclei was done with Kemechrot solution. At
least 500 cells per preparation were observed. The IC50 values (nM)
of 2 at the each experiment using the vitamin D3 lactone analogs
(72-91, 72a-91a, 72b-91b, and 72c-91c) were as follows: 6.0
(for 74, 79, 84, and 89), 6.1 (for 72c, 77c, 82c, and 87c), 6.2 (for
72b, 77b, 82b, and 87b), 6.3 (for 74a-c, 79a-c, 84a-c, and 89a-
c), 6.7 (for 73a, 75a, 78a, 80a, 83a, 85a, 88a, and 90a), 6.8 (
1.13 (for 72a and 77a), 7.7 ( 1.38 (for 72 and 77), 8.1 (for 82a
and 87a), 8.2 ( 0.85 (for 75, 80, 85, and 90), 8.4 (for 73b,c, 78b,c,
83b,c, and 88b,c), 8.6 ( 0.28 (for 73, 78, 83, and 88), 8.8 (for
75b,c, 80b,c, 85b,c, and 90b,c), 9.2 (for 87), 9.3 (for 81, 76b,c,
81b,c, 86b,c, and 91b,c), 9.5 (for 76), 10.2 ( 1.20 (for 86 and 91),
10.5 ( 0.71 (for 76a and 91a), 11.0 (for 82, 81a, and 86a). The
Supporting Information Available: General experimental
details, synthetic procedures and spectral data of 2R-substituted
vitamin D3 lactone analogs (72a-91a, 72b-91b, and 72c-92c),
details for the synthesis and structural elucidation of the CD-ring
precursors (22-31, 57-61, and 67-71) and spectral data of all
new compounds, charts of vitamin D receptor binding assay and
assay for HL-60 cell differentiation to test antagonistic activity for
all vitamin D3 lactone analogs (72-91, 72a-91a, 72b-91b, and
72c-92c). This material is available free of charge via the Internet
at http://pubs.acs.org.
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